The primary goal of this proposal is to investigate mechanisms that may be involved in the regulation of cytosolic free calcium concentration ([Ca2+]I) during myocardial ischemia and reperfusion and may be involved in the pathogenesis of ischemic injury. Changes in [Ca2+]I and [Na+]I will be monitored in perfused hearts using magnetic resonance (NMR) methods. The first specific aim of this proposal is to investigate the protective effect of activation of phosphatidylinositol-3-kinase (PI3K) on myocardial ischemia/reperfusion injury, which is involved in the protective effect of ischemic preconditioning (PC), produced by brief intermittent periods of ischemia and reperfusion prior to a sustained period of ischemia. PC minimizes ionic alterations, particularly the rise in [Ca2+]I, during the subsequent period of sustained ischemia, in parallel with a reduction in ischemic injury. Activation of PI3K occurs during PC, and is of crucial importance for the protective effect of PC. Downstream elements of the PI3K pathway are phosphorylated during PC, and the protective effect of PC can be mimicked by pharmacologically reproducing the effect of PC on a downstream component of the PI3K pathway. Differences between males and premenopausal females in susceptibility to myocardial ischemia/reperfusion injury are seen under conditions of mild calcium overload and we have preliminary evidence that PI3K is involved in the protected female phenotype, and the objective of the second specific aim is to investigate the mechanisms involved. Female hearts do not develop ischemic injury as rapidly as male hearts, and nitric oxide production is involved in the female protection, which may be related to increased PI3K activity in the female hearts and increased eNOS expression. Male/female differences are most evident under conditions of increased calcium load, and the objective of the third specific aim is to use NMR methods to measure calcium overload and to measure changes in intracellular sodium during ischemia. Calcium handling will also be examined in isolated myocytes. There are male/female differences in [Na+]I during ischemia, and our goal is to determine whether this is related to PI3K signaling, and whether the abnormalities we have seen in SR calcium handling are related to alterations in sodium homeostasis. A complete understanding of the mechanisms involved in male/female differences in susceptibility to myocardial ischemia/reperfusion injury is of great importance at the present time, in view of the controversy surrounding hormone replacement therapy for postmenopausal women, since ischemic heart disease is the number one cause of mortality in this demographic group.